Recent studies provide evidence for the sufficiency of intrinsic programs for retinal cell diversification (Cayouette et al., 2003
; Kay et al., 2005
; Mu et al., 2005
). This is consistent with the findings that wild-type retinal progenitors can turn on ath5
and differentiate into RGCs when transplanted into a lakritz
environment, suggesting that they do not need positive signals deriving from differentiated RGCs (Kay et al., 2005
). It is particularly striking that all 14 ath5:GFP progenitors we followed in the wild-type environment displayed very similar lineages, strongly suggesting the presence of stereotyped and intrinsically determined lineage programs in the retina. However, such a conclusion must be treated with caution, as we were examining these lineages only during a narrow time window, and the results of transplanting the same cells into a lakritz
mutant suggest that even in a wild-type environment, earlier ath5-positive progenitors developing in an environment devoid of previously generated RGCs might show a different lineage program.
The reproducible lineage patterns that we found among ath5
-expressing progenitors helps explain why ath5
is necessary for RGC fate but does not necessarily correlate with RGC fate (Kanekar et al., 1997
; Brown et al., 2001
; Kay et al., 2001
; Wang et al., 2001
; Yang et al., 2003
; Masai et al., 2005
). All ath5
progenitors produce RGCs, but not all ath5
-expressing progenitors become RGCs. In wild-type animals, we found that an ath5:GFP-positive progenitor divides once and only one of the two daughters (the one that expresses ath5:GFP more strongly) becomes a RGC. The other daughter usually becomes a different cell type and down-regulates ath5:GFP expression. Interestingly, we observed that this cell usually ended up in the ONL and assumed an immature photoreceptor-like morphology, suggesting that, at least during the period of our video recordings, ath5:GFP progenitors tend to be restricted to a lineage that produce one RGC and one other cell type, usually a photoreceptor.
is first expressed in post–S-phase retinal precursors (Yang et al., 2003
; Ma et al., 2004
; Masai et al., 2005
). In our study, we recorded many ath5:GFP retinal progenitors undergoing cytokinesis at the apical surface and found that ath5
mRNA colocalizes with ath5:GFP expression in M-phase progenitors. Interestingly, we found that the onset of ath5:GFP expression in dividing progenitors was almost always shortly before they entered M phase and while their nuclei were undergoing interkinetic migration toward the apical surface, suggesting that ath5
usually turns on in retinal progenitors at G2 of the final cell cycle (Miale and Sidman, 1961
). Other cases have been described in which intrinsic factors begin to be expressed in G2; for example, Prox1, a homeobox protein that is required for the determination of horizontal cells in the vertebrate retina, is initiated at G2 (Dyer et al., 2003
), but in these cases, the fates of the daughter cells have not been followed.
It has been previously argued that the distinct clones seen in the retina could have been sculpted out of a highly conserved lineage program by random cell death (Voyvodic et al., 1995
). The differences we see among ath5:GFP progenitor clones in which one of the daughter cells becomes an RGC and the other becomes a different cell type or, in the lakritz
environment, becomes another RGC are clearly not results of cell death but are caused by different lineage patterns.
Our results clearly also show that the lineage programs of ath5:GFP-expressing cells respond to negative feedback signals in the normal environment, at least during the developmental time window of this study when the first RGCs are already present. Previous studies have shown that the environment plays an important role in regulating the proportion of RGCs generated (Waid and McLoon, 1998
; Gonzalez-Hoyuela et al., 2001
). Indeed, we found that after transplantation of wild-type cells into a wild-type host, about half of ath5:GFP-positive cells are RGCs, but this percentage is increased by ~30% when the same cells are transplanted to a lakritz
mutant environment. By following individual ath5:GFP progenitors over time, we found that the environment can dramatically influence their lineage patterns, leading to a predicted increase in RGC production of ~30%. A greater number of dividing ath5:GFP progenitors give rise to two RGCs compared with one in the wild-type environment.
Recent data has shown that GDF11 is a potential negative feedback factor for RGC production (Kim et al., 2005
). In mice mutant for GDF11, the histogenetic window for ath5
expression and RGC production is extended. In these mutants, extra RGCs are generated at the expense of later born cell types. Follistatin antagonizes GDF11, and follistatin mutants show a shortened period of ath5
expression and RGC production in the retina. These results fit well with our findings of extra rounds of division and a switch from asymmetrical fate to symmetrical RGC-generating divisions in ath5 progenitors transplanted to the lakritz
Apical-basal cell divisions have been found in the vertebrate nervous system, including the retina, and some of them have been shown to be asymmetric in nature (Chenn and McConnell, 1995
; Zhong et al., 1996
; Wakamatsu et al., 1999
; Cayouette et al., 2001
; Cayouette and Raff, 2003
). Planar divisions, however, can also be asymmetric (Adams, 1996
; Gho and Schweisguth, 1998
; Wodarz and Huttner, 2003
; Kosodo et al., 2004
; Malicki, 2004
; Sun et al., 2005
). In the chick retina, cells dividing apico-basally are a small percentage of dividing cells, and this percentage does not seem to account for all of the asymmetric behavior of daughter cells (Silva et al., 2002
). Both in the zebrafish retina and neural tube, apical-basal divisions are entirely absent (Das et al., 2003
; Geldmacher-Voss et al., 2003
; Lyons et al., 2003
). Within the plane of the ventricular surface in the zebrafish retina, however, cells may divide along a more circumferential or radial axis (Das et al., 2003
). Interestingly, we found that circumferential divisions in ath5:GFP-positive progenitors tend to give rise to different or asymmetric fates. One possible explanation for this relates to the neurogenic wave, which spreads primarily in the circumferential axis (Neumann and Nuesslein-Volhard, 2000
). Consequently, circumferential divisions could produce two daughter cells that are differentially exposed to any potential signals that are associated with this wave. Differences in the intrinsic ability to respond to extrinsic signals may also account for asymmetric behavior of daughter cells. Indeed, a recent study found that unequal segregation of the EGF receptor during mitosis in dividing cortical progenitor cells plays a part in generating asymmetric behavior of the daughter cells (Sun et al., 2005
). Alternatively, these circumferential divisions may favor the unequal partitioning of an intrinsic determinant. It will be interesting to investigate which of these possibilities is at play in this system.
In this study, we found that radial divisions tend to give rise to similar or symmetric fates (in this case, two RGCs). In the Das et al. (2003)
study (Lyons et al., 2003
), all dividing cells were examined, including those early dividing cells in which presumably both daughters remained in the cell cycle. The conclusion from that study was that early progenitors might also divide primarily in a radial orientation. If that is so, the results here suggest that radial cell divisions can give rise to two progenitors at early stages or two similar postmitotic daughters at later stages.
It has always been an aspiration of scientists in this field to directly observe clone formation and to watch how different cell types arise in vivo. This time-lapse analysis is the beginning of such work, and we have used this beginning to show, for the first time, a major role for the environment in influencing the orientation of division and lineage pattern of a distinct class of retinal precursors in their final mitosis.